The present inventions relate to novel methods for purifying oligonucleotides. More specifically, the present inventions relate to novel methods for purifying oligonucleotides wherein the oligonucleotides are precipitated from solution and isolated using physical means.
Oligonucleotides and their analogs have been developed and used in molecular biology as probes, primers, linkers, adapters, and gene fragments in a variety of procedures. Oligonucleotides play a significant role, for example, in the fields of therapeutics, diagnostics, and research.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and humans. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for various disease states, including use as antiviral agents. Other mechanisms of action have also been proposed. For example, transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209), incorporated herein by reference in its entirety.
In addition to use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using, for example, biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits that assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993; each incorporated herein by reference in its entirety. Such uses include, for example, synthetic oligonucleotide probes, screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra; each incorporated herein by reference in its entirety.
Owing to the wide range of applications, oligonucleotides and their analogs have been customized to provide properties that are tailored for desired uses. Thus, a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include, but are not limited to, those designed to increase binding to a target strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, to improve the pharmacokinetic properties of the oligonucleotide, and to modulate uptake and cellular distribution of the oligonucleotide.
Modifications to naturally occurring oligonucleotides include, for example, labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include, but are not limited to, incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units.
Antisense oligonucleotides also may be modified to conjugate with lipophilic molecules. The presence of the lipophilic conjugate has been shown to improve cellular permeation of the oligonucleotide and, accordingly, improve distribution of the oligonucleotide in cells. Further, oligonucleotides conjugated with lipophilic molecules are able to enhance the free uptake of the oligonucleotides without the need for any transfection agents in cell culture studies. Conjugated oligonucleotides are also able to improve the protein binding of oligonucleotides containing phosphodiester linkages. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein each incorporated herein by reference in its entirety), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
The ability of the acylaminoethyl group to serve as a protecting group for certain phosphate diesters was first observed by Ziodrou and Schmir. Zioudrou et al., J. Amer. Chem. Soc., 85, 3258, 1963; incorporated herein by reference in its entirety. A version of this method was extended to the solid phase synthesis of oligonucleotide dimers, and oligomers with oxaphospholidine nucleoside building blocks as substitutes for conventional phosphoramidites. Iyer et al., Tetrahedron Lett., 39, 2491-2494, 1998; PCT International Publication WO/9639413, published Dec. 12, 1996; each incorporated herein by reference in its entirety. Similar methods using N-trifluoroacetyl-aminoalkanols as phosphate protecting groups has also been reported by Wilk et al., J. Org. Chem., 62, 6712-6713, 1997; incorporated herein by reference in its entirety. This deprotection is governed by a mechanism that involves removal of an N-trifluoroacetyl group followed by cyclization of aminoalkyl phosphotriesters to azacyclanes, which is accompanied by the release of the phosphodiester group.
Solid phase techniques continue to play a large role in oligonucleotidic synthetic approaches. Typically, the 3xe2x80x2-most nucleoside is anchored to a solid support that is functionalized with hydroxyl or amino residues. The additional nucleosides are subsequently added in a step-wise fashion to form the desired linkages between the 3xe2x80x2-functional group of the incoming nucleoside, and the 5xe2x80x2-hydroxyl group of the support bound nucleoside. Implicit to this step-wise assembly is the judicious choice of suitable phosphorus protecting groups. Such protecting groups serve to shield phosphorus moieties of the nucleoside base portion of the growing oligomer until such time that it is cleaved from the solid support.
After cleavage, the oligonucleotide usually must undergo treatment and processing such as, in some instances, deprotection, precipitation and isolation, in order to produce a purified oligonucleotide product. Precipitation is the process in which an oligonucleotide product in solution is treated with an anti-solvent to form an agglomerated solid in suspension. The solid product is then isolated from the liquid phase. Established methods for precipitation and drying of oligonucleotides have been well documented. Oligonucleotides can be prepared following, for example, the technique described in Maniatis"", Techniques in Molecular Biology. 
One technique for purifying oligonucleotides involves, for example, DMT-on full-length fractions that are isolated by reversed phase HPLC and pooled, precipitated in a large volume of ethanol at xe2x88x9220xc2x0 C., isolated by continuous flow high-speed centrifugation (15K), and then reconstituted in water. The 4,4xe2x80x2-dimethoxytrityl ether protecting group is removed by acidifying the aqueous oligonucleotide solution to within a range of pH 3.3 to 4.1. After the reaction is complete the solution is diluted with 3 M sodium acetate, then precipitated in ethanol at xe2x88x9220xc2x0 C., isolated by high-speed centrifugation and reconstituted in water. The aqueous oligonucleotide solution is adjusted to pH 7.0-7.4 with 1 N sodium hydroxide, precipitated in ethanol at xe2x88x9220xc2x0 C., isolated by continuous flow high speed centrifugation then reconstituted in water. The final reconstituted aqueous oligonucleotide is dried by lyophilization using a 56-hour drying cycle.
However, the above purification scheme requires the use of expensive high-speed centrifuges which are generally only able to process relatively small batches of oligonucleotide. Further, the above method requires large quantities of solvents to be cooled to xe2x88x9220xc2x0 C.
In light of the foregoing, there is a continued need for improved methods of purifying oligonucleotides. In particular, there is a need for methods of rapidly and efficiently producing a high-yield of purified oligonucleotides. The methods can preferably be performed at ambient temperature. Further, the methods preferably enable the purified oligonucleotides to be separated from solution using cost-effective isolation techniques.
The present inventions relate to novel methods for purifying oligonucleotides and for producing a high yield of purified oligonucleotide product. More specifically, the present inventions relate to novel methods for aggregating oligonucleotides and isolating the resulting aggregate. The methods of the present invention can be practiced at ambient temperature and allow the aggregates to be isolated using cost-effective physical techniques. The present inventions also relate to cost-effective downstream processing techniques for small- and large-scale operations.
The present inventions relate to methods for purifying an oligonucleotide comprising the steps of reacting the oligonucleotide with an aggregating agent and a precipitation enhancer, under conditions sufficient to form an oligonucleotide aggregate; and isolating the oligonucleotide aggregate to form an isolated oligonucleotide. In certain embodiments, the aggregating agent is an alcohol, such as methanol, ethanol, 1-propanol, isopropyl alcohol or denatured ethanol. In other embodiments, the precipitation enhancer comprises a salt, such as sodium salt (Na+), lithium salt (Li+), ammonium salt (NH4+), potassium salt (K+), magnesium salt (Mg+), cesium salt (Cs+) or zinc salt (Zn+). For example, the precipitation enhancer can be sodium acetate (NaOAc) or sodium hydroxide (NaOH).
In certain embodiments of the present inventions, the oligonucleotide is a protected oligonucleotide present in a solution. Protective groups include, but are not limited to, trimethoxytrityl, dimethoxytrityl, monomethoxytrityl, 9-phenylxanthen-9-yl, and 9-(p-methoxyphenyl)xanthen-9-yl. The oligonucleotide is preferably present in solution at a concentration of at least about 550 OD/ml, at least about 600 OD/ml, or at least about 650 OD/ml.
In other embodiments of the present inventions, the oligonucleotide is a deprotected oligonucleotide present in a solution. The oligonucleotide is preferably present in solution at a concentration of at least about 2250 OD/ml, between about 2500 OD/ml and about 7500 OD/ml, or between about 4500 OD/ml and about 6500 OD/ml.
Although the oligonucleotide can be treated with the aggregating agent at a wide range of reaction temperatures, the oligonucleotide is preferably treated with said aggregating agent at a temperature between about 15xc2x0 C. and about 25xc2x0 C., and more preferably between about 18xc2x0 C. and about 20xc2x0 C.
In certain embodiments, the oligonucleotide is treated with said precipitation enhancer prior to treating said oligonucleotide with said aggregating agent. Alternatively, the oligonucleotide can be treated with said aggregating agent prior to treating said oligonucleotide with said precipitation enhancer, or the oligonucleotide can be treated with a mixture of said precipitation enhancer and said aggregating agent.
The oligonucleotide can be present in a solution. When present in a solution, the oligonucleotide is preferably treated with an aggregating agent in a ratio of about 1 part solution to at least about 1.5 parts aggregating agent by volume, more preferably, between about 2 parts and about 4 parts aggregating agent by volume, and even more preferably, between about 2.5 parts and about 4.5 parts aggregating agent by volume.
In certain embodiments, the oligonucleotide is isolated from said solution by high- or low-speed centrifugation. Alternatively, the oligonucleotide can be isolated from said solution by gravitational settling or filtration.
In a preferred embodiment, a purified oligonucleotide is prepared by treating a first solution comprising a 5xe2x80x2-protected oligonucleotide with an aggregating agent under conditions sufficient to form a first oligonucleotide aggregate, isolating the oligonucleotide, and then dissolving the isolated oligonucleotide aggregate to form a second solution. The second solution is treated with a deprotecting reagent, to remove the 5xe2x80x2-protecting groups, with an aggregating agent and a precipitation enhancer under conditions sufficient to form a second oligonucleotide aggregate, which is isolated and dissolved to form a third solution. The third solution is treated with an aggregating agent and a precipitation enhancer under conditions sufficient to form a third oligonucleotide aggregate, which is isolated to provide a purified oligonucleotide.
In an alternate embodiment, a purified oligonucleotide is prepared by treating a first solution comprising an oligonucleotide with an aggregating agent and a precipitation enhancer under conditions sufficient to form a first oligonucleotide aggregate, isolating and dissolving the isolated first oligonucleotide aggregate to form a second solution. The second solution is treated with an aggregating agent and a precipitation enhancer under conditions sufficient to form a second oligonucleotide aggregate and isolated to produce a purified oligonucleotide.
The first solution can be prepared by acidification of HPLC effluent containing a 5xe2x80x2-protected oligonucleotide, wherein the effluent is produced by HPLC purification of a cleaved and base deblocked 5xe2x80x2-protected oligonucleotide.
In other embodiments, the resulting purified oligonucleotide is at least about 90% pure, and more preferably, at least about 98% pure. In some embodiments, the first solution is effluent obtained from high-pressure liquid chromatography of crude oligonucleotide, wherein the high-pressure liquid chromatography is performed using a column loaded with reverse phase media or strong anion exchange resin.